【摘要】：RNA干擾(RNA interference, RNAi)是近年來發(fā)現(xiàn)的在生物體內(nèi)普遍存在的一種生物學現(xiàn)象。RNA干擾技術作為重要的生物技術之一,被廣泛地應用于重大疾病的診斷與治療等領域。微小RNA(microRNAs, miRNAs)作為干擾RNA的一類,是在真核生物中發(fā)現(xiàn)的一類內(nèi)源性的、進化保守的、具有調(diào)控基因表達功能的非編碼RNA。它在包括發(fā)育、造血過程、器官形成、細胞增殖和凋亡、脂肪代謝等過程中參與調(diào)控并發(fā)揮重要作用。然而,由于miRNA在生理環(huán)境中極不穩(wěn)定、體內(nèi)半衰期短且其自身無法滲透到細胞內(nèi),亟待開發(fā)穩(wěn)定、高效、無毒的投遞體系以滿足基于miRNA的疾病治療。本文針對目前miRNA孰體在投遞過程中面臨的毒性高、投遞效率低、穩(wěn)定性差等困境,設計了一種基于納米膠囊的miRNA投遞平臺,并研究了其在RNA干擾治療中的應用。本文首先使用原位自由基聚合方法,設計并制備出了一種低毒、高效的細胞內(nèi)miRNA投遞納米膠囊。比較了聚合單體種類和比例對納米膠囊結(jié)構和表面性質(zhì)的影響,發(fā)現(xiàn)高APm/AAm比例可獲得粒徑較小、表面電荷較高的納米膠囊。在此基礎上,我們還研究了miRNA納米膠囊及商用的Lipofectamine轉(zhuǎn)染試劑與miRNA結(jié)合的復合物(Lipo/miR)在模擬生理環(huán)境中的結(jié)構穩(wěn)定性,證明較Lipo/miR,納米膠囊可更好地保護miRNA,阻止其被肝素置換和核酸酶降解。細胞實驗結(jié)果表明,納米膠囊比Lipofectamine具有更低的細胞毒性,卻可更高效地轉(zhuǎn)染miRNA。利用納米膠囊投遞反義microRN A-21 (As-miR-21),可以顯著地降低腫瘤細胞內(nèi)miR-21表達,調(diào)控PTEN/PIK-Akt信號通路抑制相關轉(zhuǎn)錄因子例如β-catenin、HIF1-α和STAT3的表達以及它們的核轉(zhuǎn)位,進而阻斷血管內(nèi)皮生長因子(VEGF)信號轉(zhuǎn)導通路,從而抑制體內(nèi)腫瘤血管增生和腫瘤生長。其次,本文針對目前miRNA在急性腦缺血治療中面臨的局部注射風險較大、靜脈注射投遞效率低等困境,設計并制備了一種可用于靜脈注射并高效投遞miRNA的納米膠囊。比較了聚合單體種類和比例對納米膠囊結(jié)構和表面性質(zhì)的影響,發(fā)現(xiàn)高單體比例可獲得粒徑更小更均一、表面電荷較高的納米膠囊；增加PEG用量可更有效地屏蔽納米膠囊表面電荷,并降低納米膠囊表面的非特異性蛋白吸附。利用動態(tài)光散射和凝膠電泳研究了納米膠囊殼層的降解行為,發(fā)現(xiàn)miRNA納米膠囊可在生理環(huán)境中保持結(jié)構穩(wěn)定,而在酸性條件下聚合物殼層發(fā)生降解從而將miRNA釋放出來。激光共聚焦和流式細胞術實驗結(jié)果證明,PEG的引入顯著降低了巨噬細胞對納米膠囊的內(nèi)吞,而星形來源的神經(jīng)膠質(zhì)細胞的內(nèi)吞效率仍維持在較高水平,這顯著降低了體內(nèi)網(wǎng)狀內(nèi)皮系統(tǒng)(RES)對納米膠囊的攝取,增強了其在腦部的富集并保證了miRNA進入細胞發(fā)揮功能。在大鼠急性腦缺血模型中,利用納米膠囊尾靜脈注射投遞具有抗凋亡和促血管新生功能的miR-21模擬物,可顯著改善模型大鼠的神經(jīng)功能缺損。本文最后針對腫瘤治療的多靶點聯(lián)合用藥要求,以納米膠囊技術為基礎,制備了同載As-miR-21和化療藥物阿霉素(DOX)的納米膠囊和包裹腫瘤抑制因子VHL蛋白的納米膠囊。MTT實驗證明了,利用納米膠囊聯(lián)合投遞DOX和AS-miR-21可協(xié)同抑制腫瘤細胞增殖。進一步的研究結(jié)果顯示,聯(lián)合投遞As-miR-21、DOX和VHL可以實現(xiàn)對腫瘤細胞內(nèi)信號通路的協(xié)同調(diào)控,實現(xiàn)對靶點因子的最大抑制。
[Abstract]:RNA interference (RNAi) is a ubiquitous biological phenomenon found in organisms in recent years. As one of the important biotechnologies, RNA interference technology is widely used in the diagnosis and treatment of major diseases and other fields. A class of endogenous, evolutionarily conserved, noncoding RNA that regulates gene expression. It is involved in regulation and plays an important role in many processes, including development, hematopoiesis, organogenesis, cell proliferation and apoptosis, and fat metabolism. However, due to the extremely unstable physiological environment, microRNAs have short half-lives and are not permeable by themselves. A stable, efficient and non-toxic delivery system is urgently needed to meet the needs of disease treatment based on microRNAs. In this paper, a microRNAs delivery platform based on nanocapsules is designed to overcome the difficulties of high toxicity, low delivery efficiency and poor stability in the delivery of microRNAs. Firstly, a low toxicity and high efficiency intracellular microRNAs delivery nanocapsules were designed and prepared by in situ free radical polymerization. The effects of the kinds and proportions of polymeric monomers on the structure and surface properties of the nanocapsules were compared. It was found that nanocapsules with small particle size and high surface charge could be obtained by high APm/AAm ratio. We also studied the structural stability of microRNA nanocapsules and commercial Lipofectamine-binding compounds (Lipo/microRNAs) in simulated physiological environments. The results showed that nanocapsules could protect microRNAs from heparin replacement and nuclease degradation better than Lipo/microRNAs. Nanocapsule delivery of antisense microRN A-21 (As-microRN A-21) significantly reduces the expression of microRN A-21 in tumor cells, and regulates the PTEN/PIK-Akt signaling pathway by inhibiting the expression of related transcription factors such as beta-catenin, HIF1-a and STAT3, as well as their nuclear translocation, thereby blocking blood translocation. Vascular endothelial growth factor (VEGF) signal transduction pathway inhibits tumor angiogenesis and tumor growth in vivo. Secondly, in view of the difficulties of high local injection risk and low intravenous delivery efficiency in the treatment of acute cerebral ischemia, we designed and prepared a kind of nanoparticles which can be used for intravenous injection and highly efficient delivery of microRNAs. Rice capsules. Comparing the effects of the types and proportions of polymeric monomers on the structure and surface properties of nanocapsules, it was found that nanocapsules with smaller and more uniform particle size and higher surface charge could be obtained with higher proportions of monomers. The degradation behavior of the nanocapsule shell was studied by dynamic light scattering and gel electrophoresis. It was found that the structure of the nanocapsule was stable in the physiological environment, and the degradation of the polymer shell under acidic conditions led to the release of the microRNAs. The results of laser confocal focusing and flow cytometry showed that the introduction of PEG significantly reduced macrophages. The uptake of nanocapsules by the reticuloendothelial system (RES) in vivo was significantly reduced, and the accumulation of microRNAs in the brain was enhanced and the function of microRNAs was ensured in the rat model of acute cerebral ischemia. The neurological deficits of the model rats were significantly improved by intravenous delivery of microRNAs-21 mimics with anti-apoptosis and angiogenesis functions. Finally, nanocapsules loaded with As-microRNAs-21 and doxorubicin (DOX) were prepared based on nanocapsule technology to meet the multi-target combination requirements of tumor therapy. Nanocapsules encapsulating tumor suppressor VHL proteins have been demonstrated by MTT experiments to synergistically inhibit the proliferation of tumor cells by using nanocapsules combined with DOX and AS-miR-21. Further studies have shown that as-miR-21, DOX and VHL can achieve synergistic regulation of signal pathways in tumor cells and maximize target factors. Inhibition.
【學位授予單位】：天津大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R943;TB383.1
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本文編號：2199591